Braunbeck, S. | bioRxiv Science

bioRxiv2021

The action of Con-ikot-ikot toxin on single AMPA-type glutamate receptors

Conotoxins are a large group of naturally occurring toxic peptides produced by the predatory sea snails of the genus Conus. Many of these toxins target ion channels, often with high specificity and affinity. As such, they have proven to be invaluable for basic research as well as acting as leads for therapeutic strategies. Con-ikot-ikot is the only conotoxin so far identified that targets AMPA-type glutamate receptors, the main mediators of excitatory neurotransmission in the vertebrate brain. Here, we describe how the toxin modifies the activity of AMPA receptors at the single-channel level. The toxin binds to the AMPA receptor with high affinity (EC50 = 5 nM) and once bound, takes minutes to wash out. As shown previously, it effectively blocks desensitization of AMPA receptors, however, compared to other desensitisation blockers, it is a poor stabiliser of the open channel because toxin-bound AMPA receptors undergo frequent, brief closures. We propose this is a direct consequence of the toxins unique binding mode to the ligand binding domains. Unlike other blockers of desensitization, which stabilise individual dimers within an AMPA receptor tetramer, the toxin immobilizes all four ligand binding domains of the tetramer. This result further emphasises that quaternary reorganization of independent LBD dimers is essential for the full activity of AMPA receptors.

neuroscience↗

bioRxiv2020

Non-selective cation permeation in an AMPA-type glutamate receptor

Fast excitatory synaptic transmission in the central nervous system relies on the AMPA-type glutamate receptor (AMPAR). This receptor incorporates a non-selective cation channel which is opened by the binding of glutamate. Although the open pore structure has recently became available from cryo-electron microscopy (Cryo-EM), the molecular mechanisms governing cation permeability in AMPA receptors are not understood. Here, we combined microsecond molecular dynamics (MD) simulations on a putative open state structure of GluA2 with electrophysiology on cloned channels to elucidate ion permeation mechanisms. Na+, K+ and Cs+ permeated at physiological rates, consistent with a structure that represents a true open state. A single major ion binding site for Na+ and K+ in the pore represents the simplest selectivity filter (SF) structure for any tetrameric cation channel of known structure. The minimal SF comprised only Q586 and Q587, and other residues on the cytoplasmic side formed a cone- shaped void that lacked major interactions with ions. We observed Cl- invasion of the upper pore, explaining anion permeation in the edited form of GluA2. A permissive architecture of the SF accommodated different alkali metals in distinct solvation states to allow rapid, non-selective cation permeation, and co-permeation by water. Simulations suggested Cs+ uses two equally populated ion binding sites in the filter and we confirmed with electrophysiology of GluA2 that Cs+ is more permeant than Na+, consistent with serial binding sites preferentially driving selectivity. Significance StatementAMPA-type glutamate receptors (AMPARs) are key actors in neurotransmission, making the final step in a relay of excitability from one brain cell to another. The receptor contains an integral ion channel, which, when opened by neurotransmitter binding, permits sodium and other cations to cross the cell membrane. We investigated permeation of sodium, potassium and caesium in an AMPAR at the atomistic level using a computational molecular dynamics approach on a structure with the ion channel pore in a presumably open state. We determined that the region selecting between cations is the simplest of any channel of this type. Distinct from ion channels that select single ion species, cations are never fully dehydrated and have only one major ion binding site in the filter. Simulations suggested two similar binding sites for caesium, and studies of AMPARs in mammalian cell membranes showed that this makes caesium more permeant than sodium.

biophysics↗